A Comparison between Cytotoxicity Induced by Two Resin Based Sealers (2Seal and AH Plus) in Saos-2 and MG-63 Cell Lines.

The aim of this study was to evaluate and compare the cytotoxicity induced by two resin-based sealers, 2Seal and AH Plus, in two osteoblast-like cell lines, MG-63 and Saos-2. Using sterile discs of both sealers in complete media, 24- and 72-h extracts were prepared. The extracts were exchanged with Saos-2 or MG-63 cell culture media at 75% confluence, and after 24 h incubation, cell viability tests were performed for each extract and cell line using MTT and trypan blue dye exclusion assays. Corresponding incubated media were used as negative control groups. For both extracts and sealers, cytotoxicity was observed in both cell lines. For Saos-2, there was no statistical difference in toxicity between the sealers for either extract (p > 0.05). For MG-63, the 2Seal 24-h extract and the AH Plus 72-h extract had greater cytotoxicity than the other extracts (p < 0.05(. Both AH Plus and 2Seal demonstrated significant cytotoxicity in these two cell lines. In contrast to 2Seal, the cytotoxicity of AH Plus in the MG-63 cell line increased with extraction time from 24 to 72 h. The AH Plus and 2Seal 24-h extracts showed different levels of cytotoxicity in the MG-63 cell line, while in the Saos-2 cell line there were no detectable differences. This may reflect higher sensitivity of the MG-63 cell line compared to Saos-2 toward cytotoxicity induced by these two sealers, or different kinetics of toxicant release from the sealers.

. Cell culture based cytotoxicity assays for medical devices and dental materials have got great approvals in the recent decades compared to exhaustive and time consuming in vivo models (4,7).
Numerous cell lines including those obtained from human periodontal fibroblasts have been used for dental materials cytotoxicity assays (8,9). Also cell lines originated from tissues other than periapical or human oral cavity (e.g. 3T3, Hela, V79…) (7,10,11) have been used in these assays. However, in order to have a better prediction on biocompatibility of tested compound, it is preferred to use cell lines with similar characteristics and phenotypes to dental and periapical tissues (12,13). Since osteoblasts play an important role in healing dental and apical tissues, we chose two osteosarcoma cell lines with human origin "Saos-2 and MG-63" (14)(15)(16).
In view of the fact that the chemical composition of different sealing materials varies from one type to another, the in vitro biocompatibilitiy results would depend on the selected method of cytotoxicity assay (6). In addition to duration of extraction, the type of cell line, and exposure method would also affect the in vitro biocompatibility results. Some materials do not release toxic substances so much but show deleterious effects when come to contact with cells or tissues. At these cases, putting set discs directly in the culture vessels would simulate the in vivo condition and more likely detect cytotoxic effects.
Gutta-percha is one of the most common used root canal filling materials so far which has a very good biocompatibility (7) but other sealing materials such as zinc oxide-eugenol cement, calcium hydroxide-based, and resin-based sealers release toxic substances and show different degrees of cytotoxicities (5,9). AH Plus as a well known epoxy resin-based sealer, has shown good properties for successful endodontic therapy including less formaldehyde release, hence lower cytotoxicity in many cell lines (8,17,18). As a relatively new introduced sealing material, 2Seal has many physico-chemical properties in common with AH Plus, including its epoxy-amine resin Complete media incubated in empty wells was used as negative control.

Sealers cytotoxicity assays
All experiments were performed in triplicates. The viability of the cells was measured after Saos-2 or MG-63 cell lines exposure to the

Statistical Analysis
The AH Plus ( Fig. 1 and 2). There were no statistically differences between 2Seal and AH Plus cytotoxicities in Saos-2 cell line with both 24 and 72 hrs extracts (Fig. 1). However, there was a higher cytotoxicity on MG-63 observed by 24 hrs extract of 2Seal which decreased in favor of AH Plus with 72 hrs extract (Fig. 2).
Results obtained by trypan blue assays showed similar pattern of decrease in cell numbers ( Fig. 3, 4)

Discussion
Evaluation of cytotoxity induced by root canal filling materials using human cell lines has been widely used in recent decades to predict and compare the new materials biocompatibilities (4, 6).
In the current study we compared cytotoxicity of two    that showed higher toxicity with 24 hrs extract ( Fig.   2, 4). However, such a difference was not statistically significant in Saos-2 cell line which might be due to the difference in sensitivity of these two cell lines to the eluted toxic compounds (12) and/or the instability of eluted toxic substances in the media. The instability could be due to the fact that formaldehyde and other volatile species might leave the media by warm incubation (22). been reported in many other studies (12,21,30) and it has been recommended to perform cytotoxicity studies on different cell lines before any discrete judgment about biocompatibility of different biomaterials (12).The difference between kinetic of toxic substances release from solidified polymers might be the reason for slight differences observed by different extraction times with MG-63 cell line. However, the difference between each cell line susceptibility to the type of elutes should not be ignored as well.